CN111647640A - Method for rapidly and accurately realizing classification of cardiac function and course of chronic heart failure - Google Patents

Abstract

The invention discloses a metabolic marker related to the diagnosis or monitoring of the course of chronic heart failure and its application, wherein the metabolic marker is xanthuric acid and/or phenylacetylglycine. The present invention proves that xanthuric acid and phenylacetylglycine can be used as metabolic markers of chronic heart failure heart function B and C, respectively, to achieve accurate classification of chronic heart failure heart function, and kynurenine 3-monooxygenase ( KMO) and aldehyde dehydrogenase family 1 member A3 (ALDH1A3) can be used as drug targets for screening prevention, mitigation and/or treatment of myocardial ischemia injury in different cardiac function classes.

Description

A rapid and accurate method for grading the course of chronic heart failure cardiac function

technical field

The invention belongs to the field of heart disease, and in particular relates to a method for quickly and accurately realizing the grading of the course of chronic heart failure cardiac function.

Background technique

With the continuous recognition and development of the disease, the American College of Cardiology (ACC) and the American Heart Association (AHC) have developed a new classification, emphasizing the development and evolution of the disease. Specific grading situation: stage A is high-risk and susceptible population of heart failure, no symptoms of heart failure, and normal left ventricular function; stage B is asymptomatic, but has developed into organic and structural heart disease, and left ventricular function is abnormal; stage C: The patient has previous or current symptoms and signs of heart failure, such as shortness of breath, decreased exercise tolerance, and fluid retention, and has underlying structural heart disease; Stage D: Intractable heart failure, which may require treatment such as heart transplantation or end-of-life care . Stages B and C are two important stages in the course of chronic heart failure.

At present, chronic heart failure is mainly diagnosed clinically by measuring the content of myocardial injury markers, such as hsCRP, BNP, hs-cTn and evaluating cardiac function. However, the determination of biochemical indicators will be affected by other physiological conditions, resulting in erroneous diagnosis results. Moreover, there are very few biomarkers that can be used for grading the stage of heart failure. Based on the above practical problems, a better cardiac function evaluation method is urgently needed to achieve accurate diagnosis of chronic heart failure. High-resolution and high-throughput metabolomics technology can provide relevant metabolic characterizations for different stages of chronic heart failure, and then select specific biomarkers for different stages of chronic heart failure, thereby helping to improve the clinical understanding of different stages of heart failure. stage diagnosis and treatment.

SUMMARY OF THE INVENTION

In view of the deficiencies of the existing problems, the first object of the present invention is to provide a metabolic marker for preparing a preparation for the diagnosis or monitoring of the course of chronic heart failure; the second object of the present invention is to provide a diagnosis or monitoring of the course of heart failure. Monitoring preparation kit; the third object of the present invention is to provide the application of a metabolic marker in the preparation or screening of drugs for the treatment of chronic heart failure; the fourth object is to provide kynurenine-3-monooxygenase ( Application of KMO) and/or aldehyde dehydrogenase family 1 member A3 (ALDH1A3) as detection targets in the preparation of preparations for diagnosis or monitoring of the course of chronic heart failure; the fifth purpose is to provide KMO and/or ALDH1A3 as therapeutic targets in The application in the preparation or screening of drugs for the treatment of chronic heart failure.

The technical scheme adopted by the present invention to solve the technical problem is:

An application of a metabolic marker in the preparation of a preparation for diagnosis or monitoring of the B and C stages of chronic heart failure, wherein the metabolic marker is xanthuric acid and/or phenylacetylglycine.

A kit for diagnosing or monitoring the disease course of B and C stages of chronic heart failure, the kit contains reagents for detecting xanthuric acid and/or phenylacetylglycine.

Application of xanthuric acid and/or phenylacetylglycine as metabolic markers in the preparation or screening of drugs for the treatment of chronic heart failure.

Preferably, the chronic heart failure is in stage B or C

Application of KMO and/or ALDH1A3 as detection targets in the diagnosis or monitoring preparations of chronic heart failure B and C stages.

The application of a reagent for detecting KMO and/or ALDH1A3 in the preparation of a preparation for diagnosing or monitoring the disease course of B and C stages of chronic heart failure.

A kit for diagnosis or monitoring of the course of chronic heart failure B and C, the kit contains reagents for the detection of KMO and/or ALDH1A3.

Application of KMO and/or ALDH1A3 as therapeutic targets in the preparation or screening of drugs for the treatment of chronic heart failure.

Preferably, the chronic heart failure is in stage B or C.

Use of substances inhibiting the expression of KMO and/or ALDH1A3 in drugs for the treatment of chronic heart failure.

Preferably, the chronic heart failure is in stage B or C.

Preferably, the substance that inhibits KMO expression is UPF-648.

More preferably, the added amount of UPF-648 is 1-2 mg/kg.

Preferably, the substance that inhibits the expression of ALDH1A3 is CM10.

More preferably, the addition amount of the CM10 is 0.32-1.28 μM.

The present findings demonstrate that xanthic acid and phenylacetylglycine can be used as metabolic markers for chronic heart failure heart function B and C grades to achieve accurate grading of chronic heart failure heart function, and KMO and ALDH1A3 can be used as screening prevention, remission and/or Drug targets for the treatment of myocardial ischemia injury in different cardiac function classes.

The invention also discloses a method for simulating a mouse model of the course of chronic heart failure. , connect the artificial ventilator; after longitudinal incision of the cortex, bluntly separate the anterior chest muscles layer by layer until the ribs are exposed, puncture the thoracic cavity with curved forceps in the intercostal space, tear the pericardium, expose the heart, and squeeze the heart by lightly pressing the thorax. out of the chest cavity; quickly use a 6-0 silk thread to ligate the myocardium 3mm below the origin of the left anterior descending coronary artery along with the myocardium that the thread passes through. The mouse model conforms to clinical chronic heart failure stage B of cardiac function at 2-3 weeks, and the mouse model conforms to clinical chronic heart failure C stage of cardiac function at 4-5 weeks.

Preferably, the parameters for connecting the artificial ventilator are: a tidal volume of 3 mL, a breathing ratio of 2:1, and a heart rate of 110.

The present invention also protects the application of the mouse model obtained by the above method in the preparation for diagnosing or monitoring the course of chronic heart failure.

The present invention also protects the application of the mouse model obtained by the above method in the preparation or screening of drugs for treating chronic heart failure.

beneficial effect

(1) The present invention uses coronary artery ligation-induced chronic heart failure model mice for the first time to simulate the cardiac function stage B of clinical chronic heart failure in the 2-3 weeks after the model, and mainly simulate the clinical chronic heart failure in the 4-5 weeks. Poor heart function stage C;

(2) The present invention also found that xanthuric acid and phenylacetylglycine were specifically elevated in the urine of clinical patients and mice with chronic heart failure in stages B and C, respectively, and KMO and ALDH1A3 could be used as xanthuric acid and phenylacetylglycine, respectively. key regulatory enzymes;

(3) The present invention also finds that inhibiting KMO and ALDH1A3 respectively can effectively relieve myocardial ischemia injury.

Description of drawings

Figure 1: Time course changes of cardiac function in chronic heart failure model mice; ^# p<0.05 and ^## p<0.01 vs. Sham group.

Figure 2: Time course changes in cardiac dimensions in a mouse model of chronic heart failure; ^# p<0.05 and ^## p<0.01 vs. Shamgroup.

Figure 3: Time course changes of cardiac organ index in chronic heart failure model mice; ^# p<0.05and ^## p<0.01vs.Shamgroup.

Figure 4: Time course changes of serum brain natriuretic peptide (BNP) and high-sensitivity C-reactive protein (hs-CRP) content in chronic heart failure model mice; where, A)BNP.B)hs-CRP. ^# p< 0.05 and ^## p<0.01 vs. Sham group.

Figure 5: Changes in myocardial histopathology and degree of fibrosis in chronic heart failure model mice.

Figure 6: Changes in the degree of edema of various organs in chronic heart failure model mice; ^# p<0.05 and ^## p<0.01vs.Shamgroup.

Figure 7: Changes in exercise tolerance in chronic heart failure model mice; ^# p<0.05 and ^## p<0.01 vs. Sham group.

Figure 8: Metabolite content determination results in mouse plasma samples; ^# p<0.05 and ^## p<0.01 vs. Sham group.

Figure 9: Measurement results of metabolite content in mouse serum samples; among them, A) 2-Arachidonoylglycerophosphocholine. B) Nicotinic acid. C) Picolinic acid ). ^# p<0.05 and ^## p<0.01 vs. Sham group.

Figure 10: Determination results of metabolites in mouse urine samples; among them, A) Xanthurenicacid.B) Phenylacetylglycine.C) 1-Methylguanosine.D) N-methyl-4-pyridone-3-carboxamide (4PY). E) Fructoselysine (Fructoselysine). F) 7-Methylguanine (7-Methylguanine). ^# p<0.05 and ^## p <0.01 vs. Sham group.

Figure 11: Metabolite content determination results in clinical plasma samples; ^# p<0.05 and ^## p<0.01 vs. normal group). *p<0.05, **p<0.01.

Figure 12: Metabolite content determination results in clinical serum samples; wherein, A) 2-arachidonoyl glycerophosphocholine. B) niacin. C) picolinic acid. ^# p<0.05 and ^## p<0.01vs. normal group.*p<0.05,**p<0.01.

Figure 13: Results of determination of metabolite content in clinical urine samples; wherein, A) xanthuric acid. B) phenylacetyl glycine. C) 1-methylguanosine. D) 7-methylguanine. E) 4PY.F ) fructose lysine. ^# p<0.05 and ^## p<0.01vs.normalgroup. *p<0.05,**p<0.01.

Figure 14: Changes in the content of xanthuric acid in each organ tissue in stage B of chronic heart failure model mice; ^# p<0.05, ^## p<0.01, sham operation group vs model group (Sham vs Model).

Figure 15: Differential genes in xanthuric acid-related metabolic pathways in B-stage heart tissue of chronic heart failure model mice were selected by transcriptomics.

Figure 16: Expression of key regulatory enzymes and proteins in xanthonic acid-related metabolic pathways in the heart tissue of B and C-stage chronic heart failure model mice; ^# p<0.05 and ^## p<0.01vs.Sham group.

Figure 17: The expression of KMO in various organs and tissues of B-stage chronic heart failure model mice; ^# p<0.05, ^## p<0.01, Sham vs Model.

Figure 18: The effect of KMO inhibitor on the content of xanthuric acid and phenylacetylglycine in the urine of B-stage chronic heart failure model mice; ^# p<0.05 and ^## p<0.01vs.Sham group.*p<0.05 and** p<0.01 vs. Model group.

Figure 19: The effect of KMO inhibitor on the pathology and fibrosis degree of heart tissue in B-stage chronic heart failure model mice.

Figure 20: The effect of KMO inhibitor on the serum heart failure-related biochemical indexes of B-stage chronic heart failure model mice; wherein, A) BNP.B) hs-CRP.C) Tumor necrosis factor alpha (TNF-α).D) Malondialdehyde (MDA).E) Nitric oxide (NO). ^# p<0.05 and ^## p<0.01vs.Sham group.*p<0.05 and **p<0.01vs.Model group.

Figure 21 : Effects of KMO inhibitors on the ultrastructure of heart tissue in B-stage chronic heart failure model mice.

Figure 22: The effect of KMO inhibitors on KMO expression in the heart tissue of B-stage chronic heart failure model mice; ^# p<0.05and ^## p<0.01vs.Sham group.*p<0.05and**p<0.01vs. Model group.

Figure 23: Effects of KMO inhibitors on the viability of H9c2 cells injured by oxygen and glucose deprivation (OGD); ^# p<0.05 and ^## p<0.01vs.Control group.*p<0.05 and **p<0.01vs.OGD group.

Figure 24: Effect of KMO inhibitor on lactate dehydrogenase (LDH) leakage rate in OGD-induced injury H9c2 cells; ^# p<0.05 and ^## p<0.01 vs. Control group.*p<0.05,**p<0.01 vs. OGD group.

Figure 25: Changes of phenylacetylglycine content in various organs and tissues of C-stage mice of chronic heart failure model. ^# p < 0.05, ^## p < 0.01, Sham vs Model.

Figure 26: Differential genes in phenylacetylglycine-related metabolic pathways in C-stage cardiac tissue of chronic heart failure model mice were selected by transcriptomics.

Figure 27: Expression of key regulatory enzyme proteins in phenylacetylglycine-related metabolic pathways in the heart tissue of B and C-stage chronic heart failure model mice; ^# p<0.05 and ^## p<0.01vs.Sham group.

Figure 28: Expression of ALDH1A3 in various organs and tissues of C-stage chronic heart failure model mice; ^# p<0.05, ^## p<0.01, Sham vs Model.

Figure 29: Effect of ALDH1A3 inhibitor on the content of phenylacetylglycine and xanthuric acid in the urine of C-stage chronic heart failure model mice; ^# p<0.05 and ^## p<0.01vs.Sham group.*p<0.05 and** p<0.01 vs. Model group.

Figure 30: The effect of ALDH1A3 inhibitor on the pathology and fibrosis degree of cardiac tissue in C-stage chronic heart failure model mice.

Figure 31: The effect of ALDH1A3 inhibitor on serum heart failure-related biochemical indexes in C-stage chronic heart failure model mice; wherein, A)BNP.B)hs-CRP.C)MDA.D)TNF-α. ^# p<0.05 and ^## p<0.01vs.Sham group.*p<0.05and**p<0.01vs.Model group.

Figure 32: Effects of ALDH1A3 inhibitors on the ultrastructure of cardiac tissue in C-stage chronic heart failure model mice.

Figure 33 : The effect of ALDH1A3 inhibitors on the expression of ALDH1A3 in the heart tissue of C-stage chronic heart failure model mice; ^# p<0.05 and ^## p<0.01vs.Sham group.*p<0.05 and **p<0.01vs. Model group.

Figure 34: Effect of ALDH1A3 inhibitor on OGD-induced injury H9c2 cell viability; ^# p<0.05 and ^## p<0.01 vs. Control group.*p<0.05 and **p<0.01 vs. OGD group.

Figure 35: Effect of ALDH1A3 inhibitor on LDH leakage rate of OGD-induced injury H9c2 cells; ^# p<0.05 and ^## p<0.01vs.Control group.*p<0.05,**p<0.01vs.OGD group.

Detailed ways

The present invention will be described in further detail below in conjunction with the embodiments. If the reagents or equipment used are not marked with the manufacturer, they are regarded as conventional products that can be purchased through the market.

Example 1. Time course of stage B and stage C in chronic heart failure model mice

experimental method

1 Laboratory animal

The experimental animals were male ICR mice, weighing 20-22 g, clean grade, provided by the Center for Comparative Medicine of Yangzhou University, and in line with the quality standards of ordinary experimental animals. The license number is SCXK (Su) 2017-0007. Mice were reared in separate cages, 5 mice per cage. The room temperature was 24°C, and the relative humidity was 40-80%. Each mouse was allowed to drink water and food freely. The experiment was started after 7 days of adaptive rearing. 100 mice were randomly divided into 2 groups: 1) sham operation group (Sham, n=10 mice); 2) model group (Model, n=90 mice).

2 Preparation of mouse heart failure model

The mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, taken in the supine position, the left chest was dehaired, disinfected with iodophor, and connected to an artificial ventilator (tidal volume 3 mL, respiratory ratio 2:1, heart rate 110). After longitudinal incision of the cortex, the anterior chest muscles were bluntly separated layer by layer until the ribs were exposed, the thoracic cavity was punctured with curved forceps in the intercostal space, the pericardium was torn, and the heart was exposed. Quickly use No. 6-0 silk thread to ligate the myocardium 3mm below the origin of the left anterior descending coronary artery along with the myocardium that the thread passes through (the sham operation group only had the thread inserted without ligation). suture. After the modeling is completed, an electrocardiogram test is performed to check whether the modeling is successful.

3 Small animal high-frequency color ultrasound (US) determination

On the 1st, 7th, 14th, 21st, 28th, 35th, 42nd, 49th, and 56th days after modeling, they were sent to the Animal Experiment Center of Nanjing Medical University for echocardiography. Mice were anesthetized by isoflurane inhalation, supine position, and the cardiac ejection fraction (EF), fractional shortening (FS), and left ventricular septal thickness (IVS) were measured using a Visual Sonics Vevo2100 high-frequency color ultrasound system for small animals. ;d), left ventricular diameter (LVID; d), left ventricular posterior wall thickness (LVPW; d), corrected left ventricular mass (LV Masscorrected), left ventricular volume (LV Vol; d), pre-ejection (PEP) and stroke volume (SV=LV Vold-LV Vols).

4. Determination of BNP and hs-CRP in serum

On the 1st, 7th, 14th, 21st, 28th, 35th, 42nd, 49th, and 56th days after modeling, blood was collected by enucleation. Double antibody sandwich enzyme-linked immunosorbent assay) to measure the content of BNP and hs-CRP in serum, the specific operation steps refer to the instruction manual of the kit.

5 Detection of cardiac organ index

On the 1st, 7th, 14th, 21st, 28th, 35th, 42nd, 49th, and 56th days after modeling, the body weight of the experimental mice was weighed and recorded. And record the wet weight of each organ, thereby obtaining each organ index=organ mass/body weight.

6 HE staining

Operation steps for HE staining: bake the paraffin sections in an oven at 60°C for 1-2 hours; the paraffin sections are dewaxed with ethanol to water with conventional xylene, stained with hematoxylin for 10 minutes, rinsed with running water to remove residual color, and differentiated with 0.7% hydrochloric acid and ethanol for several seconds. bell, rinsed with running water, slices turned blue for about 15 min, 7.95% ethanol for 30 s, 8. Alcoholic eosin staining for 30 s, I 95% ethanol for 30 s, II 95% ethanol for 30 s, I 100% ethanol for 30 s seconds, II 100% ethanol for 30 seconds, phenolic xylene for 30 seconds, (1:4 phenolic 1-xylene 4) I xylene for 30 seconds, II xylene for 30 seconds, and a neutral resin seal.

7 Masson staining

Paraffin sections were dewaxed to water; chromated or demercured; washed with tap water and distilled water in turn; stained with Harris' hematoxylin solution or Weigert's hematoxylin solution for 1-2 min; washed with running water; differentiated with 0.5% hydrochloric acid alcohol for 15s; running water Rinse for 3min; Stain with Ponceau red acid fuchsin solution for 8min; Slightly rinse with distilled water; Treat with 1% phosphomolybdic acid aqueous solution for about 5min; Do not wash with water, directly counterstain with aniline blue solution or bright green solution for 5min; Treat with 1% glacial acetic acid for 1min; Dehydrate in 95% ethanol for 5 min×2 times, and dry up the liquid with absorbent paper; 100% ethanol for 5 min×2 times, dry up the liquid with absorbent paper; clear in xylene for 5 min×2 times, dry up the liquid with absorbent paper; neutral gum cover sheet.

8 Wet-dry mass ratio of viscera

On the 1st, 7th, 14th, 21st, 28th, 35th, 42nd, 49th, and 56th days after modeling, the organs were completely taken out, the blood on the surface was wiped off with filter paper, and the wet weight of each organ was recorded. Bake in an oven at 90°C for 72 hours, and weigh the dry mass of each organ to obtain the wet/dry mass ratio.

9 Swimming test to failure

On the 1st, 7th, 14th, 21st, 28th, 35th, 42nd, 49th, and 56th days after modeling, the exhaustive swimming test was performed, and a lead block of 5% body weight was attached to the base of the tail of the mice (the mice were allowed to swim with weight and quickly reach Exhausted state), put the weight-bearing mice into a swimming box with a water depth of 30cm, a smooth inner wall and a water temperature (25±2°C) to swim, and record the exhaustive swimming time of the mice (from the start of swimming to the time when the head of the mouse is completely submerged) 8s in water can no longer surface time). Wipe off moisture after each swim and blow dry with a hair dryer. Swim to exhaustion every time.

Experimental results

1 Time course changes of cardiac function in mice with chronic heart failure

High-frequency color ultrasound was used to evaluate the changes in the heart function of the chronic heart failure model mice for 56 days. The representative M-mode echocardiogram of the mice in each group is shown in Figure 1. The overall peak and trough of the chronic heart failure model mice distance was shorter than that of the control group. Compared with the sham-operated group, the ejection fraction of the mice in the model group decreased significantly on the 14th day after coronary artery ligation; the fractional shortening continued to decrease significantly after the 7th day; the ejection pre-ejection phase was significantly prolonged on the 28th day , and in the first 14 days after modeling, the pre-ejection period showed a downward trend compared with the sham operation group; the stroke volume decreased after the first day.

2. Time course changes of cardiac dimensions in mice with chronic heart failure

The changes of cardiac dimensions in chronic heart failure model mice within 56 days were further investigated by echocardiography. Compared with the sham-operated group, the left ventricular septal thickness (Fig. 2A), left ventricular diameter (Fig. 2B) and corrected Posterior LV mass (Fig. 2C) increased significantly from days 14 to 56 after coronary ligation modeling, whereas posterior LV wall thickness began to increase significantly on day 7 (Fig. 2D).

3 Time course changes of cardiac organ index in chronic heart failure model mice

By examining the cardiac organ index of the mice in each group, the myocardial hypertrophy of the chronic heart failure model mice within 56 days was evaluated. The results are shown in Figure 3. Compared with the sham-operated group, the heart organ index of the mice in the model group gradually increased on the 1st day after coronary artery ligation modeling, and reached the highest point on the 35th day.

4 Time course changes of serum BNP and hs-CRP levels in chronic heart failure model mice

The detection results of BNP content in the serum of mice in each group are shown in Figure 4A. Compared with the sham operation group, the BNP content in the model group showed a gradual increase after coronary artery ligation on the 7th day, while hs-CRP was higher than that in the sham operation group. BNP was more sensitive and significantly increased on day 1 after modeling and gradually increased to day 56 (Fig. 4B).

5 Changes of myocardial tissue pathology and fibrosis in chronic heart failure model mice

Furthermore, HE and Masson staining were used to evaluate the changes of myocardial histopathology and fibrosis in chronic heart failure model mice within 56 days, respectively. The results are shown in Figure 5. With the passage of time, the myocardial fibrosis, hyperemia and edema infiltration of inflammatory cells in the chronic heart failure model group induced by coronary artery ligation gradually increased. Mild, while the degree of injury increased significantly on days 28-35.

6 Changes in the degree of edema of various organs in chronic heart failure model mice

Fluid retention is a typical clinical manifestation of patients with chronic heart failure. By examining the changes in the wet-dry mass ratio of 5 major organs (heart, liver, spleen, lung, and kidney) in chronic heart failure model mice within 56 days, chronic heart failure was evaluated. The degree of edema in model mice. Compared with the sham-operated group, the heart, lung and kidney tissues of the model group showed significant edema on the 35th day after coronary artery ligation, while the liver and spleen tissues also showed obvious edema on the 28th and 35th day.

7 Changes of exercise tolerance in chronic heart failure model mice

Chronic heart failure patients are often prone to different degrees of exercise tolerance decline and other clinical manifestations. Through the exhaustive swimming test, the changes in exercise tolerance in 56-day chronic heart failure model mice were further investigated. The results are shown in Figure 7. Compared with the sham operation group, the mice in the model group showed a significant decrease in exhaustive swimming time on the 28th day after coronary artery ligation modeling.

The above experimental results show that the chronic heart failure model mice induced by coronary artery ligation have developed organic and structural heart disease in the 2-3 weeks after the model, and the left ventricular function is abnormal, which is in line with clinical chronic heart failure. Cardiac function stage B; and chronic heart failure model mice developed more obvious signs and symptoms of heart failure such as decreased exercise tolerance and fluid retention on the basis of qualitative and functional lesions in the 4th to 5th week. Cardiac function stage C in clinical chronic heart failure.

Example 2. Xanthine and Phenylacetylglycine as Metabolic Markers in Chronic Heart Failure Stages B and C, respectively

experimental method

1 Laboratory animal

See Example 1.

2 clinical samples

A total of 100 subjects were included in this experiment by Jiangsu Provincial Hospital of Traditional Chinese Medicine, and their plasma, serum and urine were collected respectively. Immediately after collection, the three samples were aliquoted into 150 μL/tube and stored at -80°C. All patients were clinically divided into normal healthy group (Normal group), chronic heart failure heart function B stage group (B stage) and heart function C stage group (C stage).

3 Preparation of mouse heart failure model

See Example 1.

4 Animal sample collection

Urine: fasted and watered during collection, the mice were placed in a metabolic cage to collect urine for 24 hours, the urine was kept in ice to keep low temperature, 4000 r/min, 4 °C low temperature centrifugation for 5 min, the supernatant was taken and stored in aliquots at -80°C.

Serum: Fasting for 24 hours before collection, blood was collected by removing the eyeball method. The blood samples were left standing at room temperature for 60 min and then centrifuged at 3500 rpm for 10 min. The supernatant was collected and stored in aliquots at -80°C.

Plasma: Fasting for 24 hours before collection, blood was collected by removing the eyeball method, using trisodium citrate as an anticoagulant, the blood sample was left standing at room temperature for 10 minutes, then centrifuged at 2500 rpm for 10 minutes, and the supernatant was taken and stored in aliquots at -80°C .

5 Pretreatment of samples

Thaw the frozen samples (serum, plasma and urine) at room temperature, take 50 μL of biological samples, 10 μL of internal standard working solution, and 140 μL of methanol, and vortex and mix for 1 min. The protein was precipitated by low temperature centrifugation at 13000rpm at 4°C for 30min, 160μL of supernatant was taken, centrifuged again at 13000r/min at 4°C for 30min, 130ul of supernatant was taken, filtered with a 0.22μm organic filter, put into a sample bottle and refrigerated at 4°C for later use. machine detection.

6 Chromatographic conditions

The chromatographic column used for plasma samples was SynergiTM Fusion-RP C18 column (50×2mm i.d., 2.5μm), and the chromatographic column for serum and urine samples was TSK-GEL Amide-80 (Part No.0019696) (150×2.0mmi. d., 5 μm), the column temperature was 25°C, the autosampler temperature was 4°C, and the injection volume was 5 μL. Mobile phase composition: Phase A is an aqueous solution containing 0.1% formic acid, and phase B is acetonitrile containing 0.1% formic acid. Gradient elution conditions for plasma samples: 0-5min for 5-10%B, 5-10min for 10-50%B, 10-15min for 50-70%B, then return to initial state, equilibrate for 5min; flow rate 0.4mL /min. Gradient elution conditions for serum samples: 80-75%B for 0-3min, 75-60%B for 3-8min, 60-50%B for 8-10min, 50%B for 10-12min, then back to Initial state, equilibrated for 5min; flow rate 0.2mL/min. Gradient elution conditions for urine samples: 70-65%B for 0-3min, 65-60%B for 3-8min, 60-50%B for 8-10min, 50%B for 10-22min, then return to To the initial state, equilibrate for 5min; flow rate 0.2mL/min.

7 Mass spectrometry conditions

Electrospray ionization (ESI) was selected, positive ion mode was used for detection, and multiple reaction monitoring mode (MRM) was selected for signal acquisition technology. The capillary and Nozzle voltages were 3500V and 1500V, respectively. The auxiliary gas temperature was 350 °C, the auxiliary gas flow was 10 L/min, the sheath gas temperature was 350 °C, the sheath gas flow was 12 L/min, and the nebulizer pressure was 50 psi.

Experimental results

1 Metabolite content determination results in mouse and clinical plasma samples

Determination of plasma C18-dihydrosphingosine (C18 - Changes in the content of dihydrosphingosine); changes in the content of C18-dihydrosphingosine in normal healthy group, B and C clinical plasma samples of chronic heart failure were determined by LC-MS.

The results showed that the content of C18-dihydrosphingosine in plasma showed a decreasing trend, and the results are shown in Figure 8 and Figure 11, respectively.

2 Metabolite content determination results in mouse and clinical serum samples

Determination of 2-arachidonoylglycerophosphocholesterol in serum of sham-operated and chronic heart failure model mice on days 1, 7, 14, 21, 28, 35, 42, 49, and 56 after coronary artery ligation by LC-MS Changes in base, niacin, and picolinic acid content. The results showed that, compared with the sham-operated group, the content of 2-arachidonoylglycerophosphocholine in serum increased from day 1 to week 3 after coronary artery ligation, but decreased from week 4 to week 5. 4-5 weeks were significantly lower than 2-3 weeks (Fig. 9A); serum niacin content showed a gradual decreasing trend after coronary artery ligation, and the degree of decrease was higher in 2-3 weeks than 4-5 weeks (Fig. 9B). ); the picolinic acid content in serum showed a gradually decreasing trend after coronary artery ligation (Fig. 9C).

Changes of 2-arachidonoylglycerophosphocholine, niacin and picolinic acid in clinical serum samples of normal healthy group, B and C stages of chronic heart failure were determined by LC-MS. The results showed that, compared with the normal healthy group, only the content of 2-arachidonoylglycerophosphocholine in serum samples of chronic heart failure stage B was significantly increased, and it tended to be normal in stage C of chronic heart failure; Acid and picolinic acid levels were significantly reduced in both B and C stages of chronic heart failure.

3 Determination results of metabolite content in mouse urine samples

Determination of phenylacetylglycine and N-methyl phenylacetylglycine in urine of sham-operated and chronic heart failure model mice on days 1, 7, 14, 21, 28, 35, 42, 49, and 56 after coronary artery ligation by LC-MS Changes in the content of -4-pyridone-3-carboxamide, fructoselysine, 1-methylguanosine, 7-methylguanine and xanthuric acid. The results showed that compared with the sham operation group, the content of xanthuric acid (Fig. 10A) in urine increased significantly at 2-3 weeks after coronary artery ligation, and gradually tended to the level of sham operation at 4-5 weeks; Both acetylglycine (Fig. 10B) and 1-methylguanosine (Fig. 10C) contents did not change significantly at 2-3 weeks after coronary artery ligation, but increased significantly at 4-5 weeks; urine N-methyl- The content of 4-pyridone-3-carboxamide (Fig. 10D) was significantly increased before the 1st week after coronary ligation, but decreased significantly from 2 to 5 weeks; There was a consistently decreasing trend after coronary ligation; urinary 7-methylguanine (Fig. 10F) levels increased significantly from 2 to 5 weeks after coronary ligation.

4. Determination results of metabolite content in clinical urine samples

Determination of phenylacetylglycine, N-methyl-4-pyridone-3-carboxamide, fructoselysine, 1-methylornithine in normal healthy group, chronic heart failure stage B and C clinical urine samples by LC-MS Changes in the content of glycosides, 7-methylguanine and xanthuric acid. The results showed that compared with the normal healthy group, only the content of xanthuric acid in the urine samples of chronic heart failure B stage was significantly increased, and it tended to be normal in the C stage of chronic heart failure; Both methylguanosine and 7-methylguanine levels were not significantly changed in chronic heart failure stage B, but were significantly elevated in stage C urine samples; N-methyl-4-pyridone- The contents of 3-carboxamide and fructoselysine were significantly decreased in both B and C stages of chronic heart failure, and there was no significant difference between the two groups.

The above experimental results show that: xanthuric acid and phenylacetylglycine are specifically elevated in the urine of clinical patients and mice with chronic heart failure in stages B and C, respectively, and can be used for the diagnosis of chronic heart failure heart function B and C.

Example 3 KMO can regulate the elevation of xanthonic acid and can be used as a new target for the treatment of chronic heart failure in stage B

experimental method

1 Laboratory animal

For other details, see Example 1. The difference is that 60 mice were randomly divided into 6 groups:

1) Sham operation group (Sham, n=10): intraperitoneal injection (ip) of equal volume of sodium chloride injection after operation, starting from the 1st day after modeling, for 14 consecutive days, once a day;

2) Model group (Model-2w, n=10): intraperitoneal injection (ip) of equal volume of sodium chloride injection after surgery, starting from the 1st day after modeling, for 14 consecutive days, once a day;

3) Model group (Model-5w, n=10): intraperitoneal injection (ip) of equal volume of sodium chloride injection after surgery, starting from the 1st day after modeling, for 35 consecutive days, once a day;

4) KMO inhibitor high-dose group (UPF-648-H, n=10): intraperitoneal injection (ip) of UPF-648 solution 2 mg/kg after surgery, starting on the 1st day after modeling, for 14 consecutive days, once/day day;

5) KMO inhibitor low-dose group (UPF-648-L, n=10): intraperitoneal injection (ip) of UPF-648 solution 1 mg/kg after surgery, starting on the 1st day after modeling, once every 3 days, for a total of 14 days;

6) Metoprolol group (Met, n=10 animals): Metoprolol 5.14 mg/kg was intragastrically administered (ig) after the operation, starting on the 1st day after modeling, for 14 consecutive days.

2 cells

The H9c2(2-1) cardiomyocyte cell line was purchased from the Shanghai Chinese Academy of Sciences Cell Bank.

3 Preparation of mouse heart failure model

See Example 1.

4 LC-MS tissue sample collection

After the mice were sacrificed, the heart, liver, spleen, lung, kidney and brain were quickly dissected out, and the blood stains on the surface of each tissue were rinsed with normal saline, and the remaining water stains were blotted dry with filter paper, weighed, and added with a quantitative physiological solution. Saline (1 g of tissue plus 2 mL of normal saline) was used to make a homogenate of each tissue with a glass homogenizer, which was stored in aliquots at -80°C.

5 Pretreatment of LC-MS samples

Thaw cryopreserved tissue samples (heart, liver, spleen, lung, kidney and brain) at room temperature, take 50 μL of tissue samples, 10 μL of internal standard working solution, and 140 μL of methanol, vortex and mix for 1 min. The protein was precipitated by low temperature centrifugation at 13000rpm at 4°C for 30min, and 160μL of supernatant was taken, and centrifuged again at 13000r/min at 4°C for 30min. machine detection.

6 LC-MS chromatographic conditions

The chromatographic column used for tissue samples was TSK-GEL Amide-80 (Part No.0019696) (150×2.0mmi.d., 5μm), the column temperature was 25°C, the temperature of the autosampler was 4°C, and the injection volume was 25°C. to 5 μL. Mobile phase composition: Phase A is an aqueous solution containing 0.1% formic acid, and phase B is acetonitrile containing 0.1% formic acid. Gradient elution conditions of the sample: 70-65%B for 0-3min, 65-60%B for 3-8min, 60-50%B for 8-10min, 50%B for 10-22min, and then back to the initial stage state, equilibrated for 5min; flow rate 0.2mL/min.

7 LC-MS mass spectrometry conditions

Electrospray ionization (ESI) was selected, positive ion mode was used for detection, and multiple reaction monitoring mode (MRM) was selected for signal acquisition technology. The capillary and Nozzle voltages were 3500V and 1500V, respectively. The auxiliary gas temperature was 350 °C, the auxiliary gas flow was 10 L/min, the sheath gas temperature was 350 °C, the sheath gas flow was 12 L/min, and the nebulizer pressure was 50 psi.

8 Detection of protein expression by Western Blotting

The hearts of mice in the sham-operated group (Sham), model group (Model-2w) and model group (Model-5w) were perfused with normal saline and taken out, and the rest of the heart except for the left ventricle of the heart was removed and homogenized with 1mM PMSF-containing homogenate. BCA kit quantification. Each group of samples was loaded on a 10% SDS-PAGE gel, and the bands after transfer were incubated with the corresponding primary antibody at 4°C overnight. After washing, the protein bands on the PVDF membrane were incubated with the corresponding secondary antibody, and the ECL kit developed color. After exposure to a gel imager, the bands were analyzed, and the protein content was expressed as the relative value of the corresponding internal reference band. Western blotting was mainly used to investigate the protein expression of regulatory enzymes A-E in the heart tissue of mice in each group.

9 Immunohistochemical staining

Each organ tissue was collected, and the expression of regulatory enzymes was analyzed by immunohistochemistry. The hearts were taken out and fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm sections. Sections were hydrated with PBS and placed in 3% hydrogen peroxide solution to block peroxidase. Sections were taken out and placed in blocking solution at 37°C for 1 hour, and incubated with primary antibody (1:100) overnight at 4°C. After washing with PBS, sections were incubated with HRP-Conjugated Secondary antibody (1:200) at 37°C for 1 hour. After DAB staining, hematoxylin counterstaining and section dehydration, the slides were mounted and observed under a 200X microscope.

10. Determination of biochemical indexes in serum

Blood samples were collected by removing the eyeballs. The blood samples were left standing at room temperature for 60 min and then centrifuged at 3500 rpm for 10 min. The supernatant was collected and the serum BNP, hs-CRP, TNF-α and MDA were measured by ELISA kit (double-antibody sandwich enzyme-linked immunosorbent assay). and NO content, the specific operation steps refer to the kit instruction manual.

11 HE staining

See Example 1.

12 Masson staining

See Example 1.

13 TEM inspection

1) Fixation: Collect the treated mouse heart tissue, add 1 mL of 2.5% glutaraldehyde respectively, shake it for about 2 minutes, shake it once every 15 minutes, and then place it in a 4°C refrigerator for more than 2 hours for later use;

2) Rinsing: Rinse 3 times with 0.1M PBS buffer (pH 7.2) for 15 min each time. to remove residual glutaraldehyde;

3) Post-fixation: transfer the tissue specimen into a centrifuge tube filled with 1% osmic acid fixative and shake it for about 5 minutes to fully infiltrate the heart tissue with starvation acid, and fix for 1 hour;

4) Rinsing: Rinse 3 times with 0.1M PBS buffer (pH 7.2) for 15 min each time. Rinse once with double-distilled water, shake well for about 10 minutes, and remove PBS;

5) Block dyeing: 1% uranyl acetate dyeing solution was treated for 2h, and when the precipitate appeared, the dyeing solution was sucked off and washed with double distilled water;

6) Gradient dehydration: immerse the specimens in 50%, 70%, 80%, and 90% acetone for 15 minutes each, and soak in pure acetone twice for a total of 20 minutes;

7) Infiltration: acetone/embedding medium = 1:1, oven at 37 °C for 1 h; acetone/embedding medium = 1:4, oven at 37 °C overnight; oven in pure embedding solution at 45 °C for 1 hour;

8) Embedding polymerization: oven drying;

9) Ultrathin section;

10) Observation and photographing by transmission electron microscope.

14 Cell Culture and Grouping

14.1H9c2 Cardiomyocyte Culture

Rat cardiomyocyte cell line (H9c2(2-1)) was cultured in DMEM medium containing 10% FBS, and cultured in a 37°C, 5% CO ₂ incubator. The cells grow adherently. Passage when they are more than 80% full, discard the culture medium, add 2 mL of PBS to gently wash, discard the washing solution, add 2 mL of 0.25% trypsin solution, and digest at 37°C until the cells shrink. Round, discard the digestion solution and add medium containing 10% FBS to terminate the digestion, pipetting repeatedly, transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in the culture medium containing 10% FBS medium, seeded at 1×10 ⁵ cells/mL in a new culture flask, and cultured in a 37°C, 5% CO ₂ incubator.

14.2 Cryopreservation of H9c2 cardiomyocytes

Take the cells in the logarithmic growth phase, digest the cells with 0.25% trypsin, add DMEM medium containing 10% FBS to terminate the digestion, collect the cell suspension, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in the freezing medium , the cells in the cryopreservation solution finally reached 2×10 ⁶ cells/mL, and were distributed into cryopreservation tubes. After aseptically sealing the cryopreservation tube, immediately place it in a 4°C refrigerator for 30 minutes, then transfer it to -20°C, place it for 2 hours, and then place it in a -70°C low-temperature refrigerator overnight (16-18h), and transfer it to liquid nitrogen the next day. save in.

14.3 H9c2 cardiomyocyte recovery

Take out the cell cryovial from the liquid nitrogen tank, and immediately put it into a 37°C water bath and shake quickly, so that the cells in the cryovial are completely thawed within 1 min. Centrifuge at 1000 rpm for 5 min at room temperature, discard the supernatant, add 1 mL of DMEM complete medium to resuspend, blow evenly, transfer the cell suspension to a culture flask, add sufficient medium, and incubate at 37 °C, 5% CO ₂ incubator nourish.

14.4 Experiment grouping

Take logarithmic growth phase H9c2 rat cardiomyocytes, digest, resuspend in DMEM medium containing 10% FBS, and inoculate in 96-well plates at a seeding density of 6 × 10 ⁴ cells/mL, add 100 μL of 10 cells/mL to each well %FBS medium, placed at 37 ° C, 5% CO ₂ incubator for normal culture, after the cells entered the logarithmic growth phase, according to the following method to establish the oxygen and glucose deprivation (Oxygen and glucose deprivation, OGD) of the N ₂ hypoxia box The models are grouped as follows:

1) Normal culture group (Control): normal culture based on normal culture in a 37°C, 5% CO ₂ incubator;

2) Normal culture group+UPF-648 high-dose group (Control+UPF-648-H): after adding UPF-648 2μM, normal culture based on 37°C, 5% CO ₂ incubator;

3) Normal culture group + UPF-648 medium dose group (Control+UPF-648-M): after adding UPF-648 1 μM, normal culture was adopted based on normal culture in a 37°C, 5% CO ₂ incubator;

4) Normal culture group + UPF-648 low-dose group (Control+UPF-648-L): After adding UPF-648 0.5 μM, normal culture was used based on normal culture in a 37°C, 5% CO ₂ incubator;

5) OGD model group (OGD): the cells entering the logarithmic growth phase for the experiment were replaced with normal culture medium with a sugar-free medium saturated with a mixture of 94% N ₂ +5% CO ₂ +1% O ₂ for 1 h. The solution was then put into a 37°C constant temperature incubator with 94% N ₂ +5% CO ₂ +1% O ₂ for 6 hours of hypoxia.

6) OGD model group+UPF-648 high dose group (OGD+UPF-648-H): after adding UPF-648 2μM, at the same time with 94%N2+5% _CO2 +1% _O2 mixture saturated for 1h without The sugar medium was replaced with the normal medium, and the cells were cultured under hypoxia for 6 h.

7) OGD model group ₊ UPF-648 medium-dose group (OGD ₊ UPF _- 648-M): after adding UPF-648 1 μM, the mice The normal medium was replaced with sugar-free medium and cultured under hypoxia for 6 h.

8) OGD model group + UPF-648 low-dose group (OGD+UPF-648-L): after adding UPF-648 0.5μM, at the same time saturated with 94%N ₂ +5%CO ₂ +1%O ₂ gas mixture for 1h The normal medium was replaced by the sugar-free medium, and the cells were cultured under hypoxia for 6 h.

15 Cell viability assay

Take logarithmic growth phase H9c2 rat cardiomyocytes, after digestion, inoculate in a 96-well culture plate and culture at 37°C, 5% CO ₂ saturated humidity. After the cells of each experimental group were treated, the prepared MTT was added for incubation. The OD value of each well was measured with an enzyme-linked immunosorbent assay (measurement wavelength 570 nm, reference wavelength 650 nm). and cell viability was calculated.

Detection of LDH release in 16 cell supernatants

Take logarithmic growth phase H9c2 rat cardiomyocytes, after digestion, inoculate in a 96-well culture plate, inoculate 100 μL of cells with a cell concentration of 1 × 105 cells/mL in each well, and then culture at 37 °C and 5% CO ₂ saturated humidity for 24 h . After the cells of each experimental group were treated with drug administration or oxygen-glucose deprivation, the supernatant of the culture medium was taken and measured according to the instructions of the LDH kit.

Experimental results

1 Changes of xanthuric acid content in various organs and tissues in B stage of chronic heart failure model mice

The content of xanthuric acid in heart, liver, spleen, lung, kidney and brain tissue of sham-operated and B-stage chronic heart failure model mice was quantitatively detected by LC-MS. The specific results are shown in Figure 14. Compared with the sham operation group, the content of xanthuric acid in the heart tissue of the model mice with chronic heart failure stage B was significantly increased, while the content of xanthuric acid in the liver, kidney and spleen tissue was significantly lower than that in the sham group. Surgery, stage B model mice and sham-operated groups showed no significant differences in lung and brain tissue.

2. Selection of differential genes in xanthonic acid-related metabolic pathways in B-stage cardiac tissue of chronic heart failure model mice by transcriptomics

Xanthuric acid is mainly involved in the tryptophan metabolic pathway, as shown in Figure 15A. Based on previous transcriptomic results, the key regulatory enzyme genes involved in xanthonic acid-related metabolic pathways include HemK methyltransferase family member 1 (HEMK1), kynurenine aminotransferase 1 (KAT1) and KMO. The RNA levels of genes in the heart tissue of the sham-operated group and the B-stage chronic heart failure model mice are shown in Figure 15B-D. Compared with the sham-operated group, HEMK1 and KAT1 in the B-stage chronic heart failure model mouse heart tissue The RNA levels were significantly decreased in all of them, while the KMO was significantly increased.

3 Expressions of key regulatory enzymes and proteins in Xanthurenic acid-related metabolic pathways in the heart tissue of mice with chronic heart failure in stages B and C

Western Blotting was used to detect the key regulatory enzyme proteins KAT1, kynurenine aminotransferase 3 (KAT3), KMO, canine and Xanthurenic acid-related metabolic pathways in the heart tissue of the sham-operated group, B and C-stage chronic heart failure model mice, respectively. Expression of ureaminidase (KYNU) and HEMK1. The specific results are shown in Figure 16. Compared with the sham operation, the expressions of KAT3, KMO and KYNU in the heart tissue of the B-stage chronic heart failure model mice were significantly increased, and there was no significant difference between the C-stage expression and the sham operation group. The expression of HEMK1 was significantly decreased, but it also decreased in the C stage. In addition, KAT1 did not change significantly in the B stage until it was significantly elevated in the C stage.

4 Expression of KMO in various organs and tissues of B-stage chronic heart failure model mice

The expression of KMO in the heart, liver, spleen, lung, kidney and brain tissues of mice in the sham-operated group and the B-stage chronic heart failure model group was investigated by immunohistochemistry. The results are shown in Figure 17. The expression of KMO in the heart tissue of the mice in the chronic heart failure model group was significantly increased, and the expression level of KMO in the remaining tissues was not significantly different from that in the sham operation group.

5 Effects of KMO inhibitors on the content of xanthuric acid and phenylacetylglycine in urine of B-stage chronic heart failure model mice

LC-MS was used to investigate whether the KMO inhibitor UPF-648 can effectively inhibit the increase of xanthuric acid in the urine of B-stage chronic heart failure model mice. The results are shown in Figure 18A. The doses of 2 mg/kg and 1 mg/kg UPF-648 can effectively inhibit the increase of xanthuric acid content in the urine of B-stage chronic heart failure model mice, and the positive drug metoprolol has no inhibitory effect on the increase of its content. In addition, the effect of the KMO inhibitor UPF-648 on the content of phenylacetylglycine in the urine of mice in each group was also investigated. The results are shown in Figure 18B. Phenylacetylglycine levels in urine were not affected. .

6 Effects of KMO inhibitors on the pathology and fibrosis degree of heart tissue in B-stage chronic heart failure model mice

The changes of myocardial histopathology and fibrosis in stage B chronic heart failure model mice after administration of KMO inhibitor UPF-648 were evaluated by HE and Masson staining, respectively. The results are shown in Figure 19. The B-stage chronic heart failure mice showed obvious myocardial fibrosis, congestion, edema, and inflammatory cell infiltration. Both 2 mg/kg and 1 mg/kg of UPF-648 can effectively improve the pathology and fibrosis of the model mice, and the high dose of UPF-648 has the best improvement effect.

7 Effects of KMO inhibitors on serum heart failure-related biochemical indexes in B-stage chronic heart failure model mice

The effects and changes of serum heart failure-related biochemical indexes in B-stage chronic heart failure model mice after administration of KMO inhibitor UPF-648 were investigated by ELISA kit, mainly including BNP, hs-CRP, TNF-α, MDA and NO . The results are shown in Figure 20. Compared with the sham-operated group, the serum central failure biochemical indexes of the B-stage chronic heart failure mice were significantly increased, indicating that the modeling was successful. Compared with the model group, it was found that UPF-648 at a dose of 2 mg/kg had a better regulation effect on heart failure biochemical indicators such as BNP, hs-CRP, TNF-α, MDA and NO in the serum of B-stage chronic heart failure mice. , and UPF-648 with a dose of 1 mg/kg showed better improvement effects except BNP, other biochemical indicators hs-CRP, TNF-α, MDA and NO.

8 Effects of KMO inhibitors on the ultrastructure of heart tissue in B-stage chronic heart failure model mice

The results of transmission electron microscopy are shown in Figure 21. The myocardial cells of the mice in the sham-operated group are arranged neatly, the muscle fibers are arranged regularly, the intercellular connections are clear, uninterrupted and the structure is relatively complete; there are neatly arranged myofibrils in the cytoplasm; Contains a large number of mitochondria and is structurally intact; the nucleus is central and structurally intact. Compared with the sham-operated group, the myofibrils of the cardiomyocytes of the mice in the B-stage chronic heart failure model group were disorderly arranged, separated, broken and dissolved, and the intercellular connections were blurred. The number of mitochondria increased and accumulated. Numerous vacuoles, indicating successful modeling. High and low doses (1mg/kg and 2mg/kg) of KMO inhibitor UPF-648 and metoprolol positive drug group showed less myocardial fibrosis than the model group, the myofibril arrangement of cardiomyocytes returned to normal, and the morphology of nuclei was also improved. tend to be normal.

9 Effects of KMO inhibitors on KMO expression in heart tissue of B-stage chronic heart failure model mice

The effect of administration of KMO inhibitor UPF-648 on the expression of KMO in the heart tissue of the B-stage chronic heart failure model mice was investigated by immunohistochemical technology. The results are shown in Figure 22. Compared with the sham operation, the B-stage chronic heart failure model The KMO expression level in the mouse heart tissue was significantly increased, which was consistent with the previous research results, and after administration of UPF-648, both high and low dose groups showed better inhibition of KMO expression.

The effect of 10 KMO inhibitors on the viability of OGD-induced injury H9c2 cells

H9c2 cardiomyocytes were cultured by oxygen and glucose deprivation to simulate in vivo ischemic environment, and the effect of KMO inhibitor UPF-648 on the viability of H9c2 cells induced by OGD was investigated by MTT assay. The results are shown in Figure 23. Compared with the blank group, hypoxia of H9c2 cardiomyocytes in the OGD group for 6 h significantly decreased the cell viability, indicating that the modeling was successful. Compared with the OGD group, administration of the KMO inhibitor UPF-648 (1μM and 2μM) significantly improved the cell viability of the model, and had no effect on the cell viability of the blank group cells.

11 Effects of KMO inhibitors on LDH leakage rate in OGD-induced injury H9c2 cells

LDH kit was used to investigate the effect of KMO inhibitor UPF-648 on the LDH leakage rate of OGD-induced injury H9c2 cells. The results are shown in Figure 24. OGD stimulation significantly increased the LDH leakage rate of H9c2 cells. The LDH release of H9c2 cells was significantly inhibited, indicating that UPF-648 could alleviate the cell membrane damage caused by OGD, and had no significant effect on the LDH leakage rate of normal cells.

The above experimental results show that the expression of kynurenine 3-monooxygenase (KMO) is specifically increased in the heart tissue of the B-stage chronic heart failure mice, which is the cause of xanthuric acid in the urine of the B-stage chronic heart failure mice. Key regulatory enzymes with increased specificity. In addition, myocardial ischemic injury can be effectively alleviated by inhibiting KMO.

Example 4. ALDH1A3 can regulate the elevation of phenylacetylglycine and can be used as a new target for C-stage treatment of chronic heart failure

experimental method

1 Laboratory animal

For other details, see Example 1. The difference is that 60 mice were randomly divided into 6 groups:

1) Sham operation group (Sham, n=10): intraperitoneal injection (ip) of equal volume of sodium chloride injection after operation, starting from the 1st day after modeling, for 35 consecutive days, once a day;

2) Model group (Model-2w, n=10): intraperitoneal injection (ip) of equal volume of sodium chloride injection after surgery, starting from the 1st day after modeling, for 14 consecutive days, once a day;

3) Model group (Model-5w, n=10): intraperitoneal injection (ip) of equal volume of sodium chloride injection after surgery, starting from the 1st day after modeling, for 35 consecutive days, once a day;

4) ALDH1A3 inhibitor high-dose group (CM 10-H, n=10 animals): intraperitoneal injection (ip) of CM 10 solution 2 mg/kg after surgery, starting on the 1st day after modeling, once every 3 days, for a total of 35 days ;

5) ALDH1A3 inhibitor low-dose group (CM 10-L, n=10 animals): intraperitoneal injection (ip) of CM 10 solution 1 mg/kg after surgery, starting on the 1st day after modeling, once every 3 days, for a total of 35 days ;

6) Metoprolol group (Met, n=10 animals): Metoprolol 5.14 mg/kg was intragastrically administered (ig) after the operation, starting on the 1st day after modeling, for 35 consecutive days, once a day.

2 Preparation of mouse heart failure model

See Example 1.

3 LC-MS tissue sample collection

See Example 3.

4 Pretreatment of LC-MS samples

See Example 3.

5 LC-MS chromatographic conditions

See Example 3.

6 LC-MS mass spectrometry conditions

See Example 3.

7 Detection of protein expression by Western Blotting

The cardiac homogenates of the sham-operated group (Sham), the model group (Model-2w) and the model group (Model-5w) were taken for Western blotting detection, and the protein expressions of ALDH1A3, ALDH3B1 and ALDH3B2 in the heart tissue of the mice in each group were mainly investigated. Happening. See 3.1.8 for the rest of the steps.

8 Immunohistochemical staining

Each organ tissue was collected, and the expression of ALDH1A3 was analyzed by immunohistochemistry. See 3.1.9 for the rest of the steps.

9 Determination of Biochemical Index Content in Serum

The blood samples were collected by removing the eyeballs. The blood samples were left standing at room temperature for 60 min and then centrifuged at 3500 rpm for 10 min. The supernatant was collected, and the serum BNP, hs-CRP, TNF-α and MDA were measured by ELISA kit (double-antibody sandwich enzyme-linked immunosorbent assay). For the specific operation steps, please refer to the instruction manual of the kit.

10 HE staining

See Example 1.

11 Masson staining

See Example 1.

12 TEM inspection

See Example 3.

13 Cell Culture and Grouping

13.1 Culture of H9c2 cardiomyocytes

See Example 3.

13.2 Cryopreservation of H9c2 cardiomyocytes

See Example 3.

13.3 H9c2 cardiomyocyte recovery

See Example 3.

13.4 Experiment grouping

Take logarithmic growth phase H9c2 rat cardiomyocytes, digested, resuspended in DMEM medium containing 10% FBS, and seeded in 96-well plates at a seeding density of 6 × 104 cells/mL, adding 100 μL containing 10% FBS to each well The culture medium of FBS was placed in a 37°C, 5% CO ₂ incubator for normal culture. After the cells entered the logarithmic growth phase, the N2 hypoxia box OGD model was established according to the following method. The specific grouping conditions are as follows:

1) Normal culture group (Control): normal culture based on normal culture in a 37°C, 5% CO ₂ incubator;

2) Normal culture group + CM 10 high-dose group (Control+UPF-648-H): after adding CM 10 1.28 μM, normal culture was used based on normal culture in a 37°C, 5% CO ₂ incubator;

3) Normal culture group + CM 10 medium dose group (Control+UPF-648-M): After adding CM 10 0.64 μM, normal culture was used based on normal culture in a 37°C, 5% CO ₂ incubator;

4) Normal culture group + CM 10 low-dose group (Control+UPF-648-L): after adding CM 10 0.32 μM, normal culture was used based on normal culture in a 37°C, 5% CO ₂ incubator;

5) OGD model group (OGD): the cells entering the logarithmic growth phase for the experiment were replaced with normal culture medium with a sugar-free medium saturated with a mixture of 94% N ₂ +5% CO ₂ +1% O ₂ for 1 h. The solution was then put into a 37°C constant temperature incubator with 94% N ₂ +5% CO ₂ +1% O ₂ , and cultured under hypoxia for 6h.

6) OGD model group + CM 10 high-dose group (OGD + UPF-648-H): after adding CM 10 1.28 μM, and at the same time saturated with 94% N ₂ +5% CO ₂ +1% O ₂ mixture for 1h The sugar medium was replaced with the normal medium, and the cells were cultured under hypoxia for 6 h.

7) OGD model group + CM 10 medium dose group (OGD + UPF-648-M): after adding CM 10 0.64 μM, at the same time saturated with 94% N ₂ +5% CO ₂ +1% O ₂ mixture for 1h The sugar medium was replaced with the normal medium, and the cells were cultured under hypoxia for 6 h.

8) OGD model group + CM 10 low-dose group (OGD + UPF-648-L): after adding CM 10 0.32 μM, at the same time saturated with 94% N ₂ +5% CO ₂ +1% O ₂ mixture for 1h The sugar medium was replaced with the normal medium, and the cells were cultured under hypoxia for 6 h.

14 Cell viability assay

See Example 3.

Detection of LDH release in 15 cell supernatants

See Example 3.

Experimental results

1 Changes of phenylacetylglycine content in various organs and tissues of C-stage mice with chronic heart failure

The content of phenylacetylglycine in heart, liver, spleen, lung, kidney and brain tissues of sham-operated and C-stage chronic heart failure model mice was quantitatively detected by LC-MS. The specific results are shown in Figure 25. Compared with the sham-operated group, the content of phenylacetylglycine in the central and lung tissues of the model mice at the C-phase of chronic heart failure was significantly increased, while the content of phenylacetylglycine in the liver and spleen tissues was significantly lower. For sham surgery, there were no significant differences in kidney and brain tissue between the C-stage model mice and the sham-operated group.

2. Selection of differential genes in phenylacetylglycine-related metabolic pathways in C-stage cardiac tissue of chronic heart failure model mice by transcriptomics

Phenylacetylglycine is mainly involved in the phenylalanine metabolic pathway, as shown in Figure 26A. Based on previous transcriptomic results, the key regulatory enzyme genes involved in phenylacetylglycine-related metabolic pathways include ALDH1A3, ALDH3B1, ALDH3B1, and ALDH3B2. The RNA levels of the selected differential genes in the heart tissue of the sham-operated group and C-stage chronic heart failure model mice are shown in Figure 26B-D. Compared with the sham-operated group, ALDH1A3, ALDH3B1 and ALDH3B2 in the C-stage chronic heart failure The RNA levels in the heart tissue of the model mice were significantly reduced.

3 Expression of key regulatory enzymes and proteins in phenylacetylglycine-related metabolic pathways in the heart tissue of mice with chronic heart failure in stages B and C

Western Blotting was used to detect the expression of key regulatory enzymes ALDH1A3, ALDH3B1 and ALDH3B2 in the cardiac tissue of sham-operated group, B and C-stage chronic heart failure model mice respectively. The specific results are shown in Figure 27. Compared with the sham-operated group, only the expression of ALDH1A3 in the heart tissue of the C-stage chronic heart failure model mice was significantly increased, while the B-stage expression was not significantly different from the sham-operated group. The expression of ALDH3B1 and ALDH3B2 in the hearts of B- and C-stage chronic heart failure model mice was not different from that of sham.

4 Expression of ALDH1A3 in various organs and tissues of C-stage chronic heart failure model mice

The expression of ALDH1A3 in the heart, liver, spleen, lung, kidney and brain tissues of mice in the sham-operated group and the C-stage chronic heart failure model group was investigated by immunohistochemistry. The results are shown in Figure 28. The expression of ALDH1A3 in the heart tissue of the chronic heart failure model group was significantly increased, and the expression level of ALDH1A3 in the rest of the tissue was not significantly different from the sham operation group.

5 The effect of ALDH1A3 inhibitor on the content of phenylacetylglycine and xanthuric acid in the urine of C-stage chronic heart failure model mice

LC-MS was used to investigate whether the ALDH1A3 inhibitor CM10 could effectively inhibit the increase of phenylacetylglycine in the urine of C-stage chronic heart failure model mice. The results are shown in Figure 29A. The doses of 2 mg/kg and 1 mg/kg CM 10 can effectively inhibit the increase of phenylacetylglycine in the urine of C-stage chronic heart failure model mice, and the positive drug metoprolol also showed a significant inhibitory effect on the increase of its content. In addition, the effect of the ALDH1A3 inhibitor CM 10 on the content of xanthuric acid in the urine of the mice in each group was also investigated.

6 The effect of ALDH1A3 inhibitor on the pathology and fibrosis of the heart of C-stage chronic heart failure model mice

The changes of myocardial histopathology and fibrosis degree of C-stage chronic heart failure model mice after administration of ALDH1A3 inhibitor CM 10 were evaluated by HE and Masson staining, respectively. The results are shown in Figure 30. The mice in the C-stage chronic heart failure model group had obvious myocardial fibrosis, congestion, edema, and inflammatory cell infiltration. Both 2 mg/kg and 1 mg/kg of CM 10 can effectively improve the pathology and fibrosis degree of model mice, and the high dose of CM 10 has the best improvement effect.

7 Effects of ALDH1A3 inhibitor on serum heart failure-related biochemical indexes in C-stage chronic heart failure model mice

The effects and changes of serum heart failure-related biochemical indexes in C-stage chronic heart failure model mice after administration of ALDH1A3 inhibitor CM 10 were investigated by ELISA kit, including BNP, hs-CRP, TNF-α and MDA. The results are shown in Figure 31. Compared with the sham operation group, the serum central failure biochemical indexes of the C-stage chronic heart failure mice were significantly increased, indicating that the modeling was successful. Compared with the model group, it was found that CM 10 at doses of 2 mg/kg and 1 mg/kg could better regulate the heart failure biochemical indicators such as BNP, hs-CRP, TNF-α and MDA in the serum of C-stage chronic heart failure mice. effect.

8 Effects of ALDH1A3 inhibitor on ultrastructure of heart tissue in C-stage chronic heart failure model mice

The results of transmission electron microscopy are shown in Figure 32. The myocardial cells of the mice in the sham operation group are arranged neatly, the muscle fibers are arranged regularly, the intercellular connections are clear, uninterrupted, and the structure is relatively complete; there are neatly arranged myofibrils in the cytoplasm; Contains a large number of mitochondria and is structurally intact; the nucleus is central and structurally intact. Compared with the sham-operated group, the myofibrils of the cardiomyocytes in the C-stage chronic heart failure model group were disorderly arranged, separated, broken and dissolved, and the intercellular connections were blurred. Numerous vacuoles, indicating successful modeling. High and low doses (1mg/kg and 2mg/kg) of ALDH1A3 inhibitor CM 10 and metoprolol positive drug group showed less myocardial fibrosis than the model group, the myofibril arrangement of cardiomyocytes returned to normal, and the morphology of nuclei tended to be similar. to normal.

9 Effects of ALDH1A3 inhibitors on the expression of ALDH1A3 in cardiac tissue of C-stage chronic heart failure model mice

The effect of administration of ALDH1A3 inhibitor CM 10 on the expression of ALDH1A3 in the heart tissue of the C-stage chronic heart failure model mice was investigated by immunohistochemistry. The results are shown in Figure 33. Compared with the sham operation, the C-stage chronic heart failure model was smaller The expression level of ALDH1A3 in rat heart tissue was significantly increased. After administration of CM 10, both high and low dose groups showed a better inhibitory effect on the expression of ALDH1A3.

10 Effects of ALDH1A3 inhibitor on the viability of OGD-induced injury H9c2 cells

H9c2 cardiomyocytes were cultured in an oxygen-glucose deprivation model to simulate in vivo ischemic environment, and the effect of ALDH1A3 inhibitor CM 10 on the viability of OGD-induced H9c2 cells was investigated by MTT assay. The results are shown in Figure 34. Compared with the blank group, hypoxia of H9c2 cardiomyocytes in the OGD group for 6 h significantly decreased the cell viability, indicating that the modeling was successful. Compared with the OGD group, administration of the ALDH1A3 inhibitor CM 10 (1 μM and 2 μM) significantly improved the cell viability of the model, and had no effect on the cell viability of the blank group.

11 Effects of ALDH1A3 inhibitor on LDH leakage rate of OGD-induced injury H9c2 cells

The effect of ALDH1A3 inhibitor CM 10 on the LDH leakage rate of OGD-induced injury H9c2 cells was investigated by LDH kit. The results are shown in Figure 35. OGD stimulation significantly increased the LDH leakage rate of H9c2 cells at doses of 0.32 μM, 0.64 μM and 1.28 μM. CM 10 at μM significantly inhibited the release of LDH in H9c2 cells caused by OGD, indicating that UPF-648 could alleviate the cell membrane damage caused by OGD, and CM 10 at 1.28 μM also had a significant inhibitory effect on the leakage rate of LDH in normal cells.

The above experimental results show that the expression of aldehyde dehydrogenase family 1 member A3 (ALDH1A3) is specifically increased in the cardiac tissue of C-stage chronic heart failure mice, which is responsible for the specificity of phenylacetylglycine in the urine of C-stage chronic heart failure mice. Key regulatory enzymes for sexual elevation. In addition, myocardial ischemia injury can be effectively alleviated by inhibiting ALDH1A3.

The protection content of the present invention is not limited to the above embodiments. Variations and advantages that can occur to those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the appended claims are the scope of protection.

Claims (10)

1. The application of a metabolic marker in the preparation of a preparation for diagnosing or monitoring the course of chronic heart failure B and C stages, wherein the metabolic marker is xanthuric acid and/or phenylacetylglycine. 2 . A kit for diagnosing or monitoring the course of chronic heart failure in stages B and C, characterized in that the kit contains a reagent for detecting the metabolic marker of claim 1 . 3. The application of the metabolic marker of claim 1 in the preparation or screening of drugs for the treatment of chronic heart failure B and C stages. 4. The application of KMO and/or ALDH1A3 as a detection target in the diagnosis or monitoring preparation of chronic heart failure B and C stages. 5. The application of the reagent for detecting KMO and/or ALDH1A3 in the preparation of a preparation for diagnosis or monitoring of the course of chronic heart failure B and C stages. 6. A kit for diagnosing or monitoring the course of chronic heart failure in stages B and C, characterized in that the kit contains reagents for detecting KMO and/or ALDH1A3. 7. Application of KMO and/or ALDH1A3 as therapeutic targets in the preparation or screening of drugs for the treatment of chronic heart failure. 8. Use of a substance that inhibits the expression of KMO and/or ALDH1A3 in a drug for the treatment of chronic heart failure. 9 . The use according to claim 8 , wherein the substance that inhibits KMO expression is UPF-648. 10 . The use according to claim 8, wherein the substance that inhibits the expression of ALDH1A3 is CM10.

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